Alzheimer's disease (AD) is a neurological disorder that disproportionately affects the population over 65 years of age. Incidence of the disease increases from less than 1% at age 60-65, to 5% at age 75, to as high as 47% at age 85. As a result, 60% to 80% of all cases of dementia in persons over age 65 are caused by AD. Afflicted individuals exhibit impaired cognitive function and memory. Neither a suitable diagnostic procedure nor an effective therapeutic treatment exists for AD. Positive identification of AD requires biopsy or autopsy of the brain.
Although the etiology of AD is unknown, genetic, immunological and environmental factors have been implicated in the development of AD. Distinguishing features of AD include the presence of senile plaques as well as, neurofibrillary tangles and extensive neuronal loss in the neocortex, hippocampus and associated structures. Senile plaques consist of extracellular deposits containing a .beta.-amyloid core surrounded by a halo of dystrophic neurites, glia and astrocytes. .beta.-amyloid deposits are present in neocortex blood vessel walls. The major component of senile plaques is a 4 kDa peptide referred to as A.beta., that is proteolytically cleaved from a larger 120 kDa amyloid precursor protein (APP). Other components of the plaques include ubiquitin, amyloid P, Apo E, interleukin-1, and .alpha.-1-antichymotrypsin.
In addition to biochemical evidence supporting A.beta. involvement in AD, there are strong genetic data which suggest a link between APP and AD. A clue to the location of a gene involved in AD comes from analysis of Down syndrome patients; in these patients trisomy of chromosome 21 is responsible for the early onset of AD. Karyotype analysis of Down syndrome patients mapped the gene involved to the upper portion of the long arm of chromosome 21. The region involved encodes several genes, including the APP gene. The early onset (.about.age 35) of AD in Down syndrome patients suggests that an increase in the gene dosage of the responsible marker(s) on the long arm of chromosome 21 may contribute to the neuropathology noticed in most AD patients.
Although the majority of AD cases appear sporadic, several cases of early onset familial AD (FAD) have been reported. Genetic analysis of FAD families has established that the disorder is inherited as a dominant autosomal gene defect, which maps to the long arm of chromosome 21 and is closely linked to the APP gene. These findings are consistent with genetic data obtained from the analysis of Down syndrome patients. Several FAD families have also been identified in which an early onset of AD is strictly correlated with the presence of a mutation in exon 17 of the APP gene at amino acid 717 (Val-Ile). This mutation within the transmembrane spanning domain of the APP cosegregates with FAD. Since the families afflicted with APP717 FAD are of different ethnic origins (English, Japanese and Canadian), evidence for the involvement of the FAD gene in these cases of AD is strong. The mutation is absent from control individuals, in sporadic AD patients, in Down syndrome patients, in late onset familial AD, and also in most other cases of early onset FAD. Several additional mutations in the APP gene have been identified that can explain the occurrence of AD in other FAD families. The genetic evidence in the five distinct early onset APP717 FAD families strongly supports the hypothesis that the APP717 gene in these FAD families is directly positioned in the pathway of AD progression.
The APP gene is approximately 400 kb in length and encodes a glycosylated, transmembrane protein which may be involved in cell-cell interaction. The APP gene has at least 18 exons that create at least 5 distinct APP transcripts by alternative splicing. The predominant transcripts encode proteins of 695, 751 and 770 amino acids (these major forms of APP are referred as APP695, APP751 and APP770, respectively). Transcripts for APP695 are enriched in the brain. Transcripts encoding APP751 and APP770 mRNA species predominate in peripheral tissues. All three isoforms contain the 42 amino acid A.beta. domain. APP isoforms 751 and 770 contain an additional 56 amino acid insert encoding the Kuinitz type serine protease inhibitor (KPI). APP is proteolytically metabolized by at least two pathways. One pathway involves an .alpha.-secretase cleavage site positioned between Lys 16 and Leu 17 of A.beta. domain; proteolytic cleavage at this site precludes the formation of an amyloidogenic A.beta. entity. The second pathway produces intact, amyloidogenic A.beta. (39-42 amino acids) by proteolytic cleavages at the .beta.- and .gamma.-secretase cleavage sites of the full-length APP molecule.
The A.beta. laden senile deposits seen in AD patients are also found in aged humans and other aged mammals including non-human primates, polar bears and dogs. However, other aged mammals, such as laboratory rats and mice, do not normally develop A.beta. deposits. This could be due to the fact that the three amino acid differences present in the .beta.-amyloid sequence between human and mouse APP prevents mouse A.beta. from forming plaques. The lack of a cost-effective, experimental animal model mimicking human pathogenesis hinders the understanding AD neuropathology and developing therapeutics against AD.
Transgenic technology may offer a suitable alternative to this problem. Addition of a gene construct directing high levels of human APP or its components to key regions in the murine central nervous system may cause neuropathological changes resembling the AD phenotype. Attempts to express human amyloid precursor protein segments or the full-length wild type protein in transgenic animals have been successful. Numerous reports exist outlining expression of different wildtype, full-length and truncated APP cDNA isoforms in mouse (Kammesheidt et al., (1992) Proc. Natl. Acad. Sci., 89, 10857-10861; Sandhu et al., (1991) J. Biol. Chem., 266, 21331-21334; Quon, et al., (1991) Nature, 352, 239-241; Wirak et al., (1991) Science, 253, 323-325; Kawabata et al., (1991) Nature 3, 476-478; Patent, International publication number WO93/02189, Neve, R., Inventor). However, these previous attempts to generate transgenic mouse models for AD have essentially failed. This failure could have been caused by the presence of the endogenous mouse APP gene in the transgenic mouse, which "protects" the human .beta.A4 from depositing in the mouse brain.
Accordingly, it is an object of the present invention to provide a transgenic mouse which does not express mouse amyloid precursor protein. The transgenic mice of the present invention are useful in the determination of the in vivo function of APP and the .beta.-amyloid peptide in the central nervous system and in other tissues. These mice are being bred with transgenic mice expressing the human APP FAD with the aim of producing a strain of mice in which the only APP produced is of human origin.
The precise roles of APP in AD is not fully understood at this time. Due to the biological importance of APP in AD and other neurological disorders, the APP gene is an important target for embryonic stem (ES) cell manipulation.
The generation of APP deficient transgenic mice would aid in defining the normal role(s) of APP, and allow an animal model of APP deficiency to be used in the design and assessment of various approaches to modulating APP activity. Such APP modified transgenic mice can also be used as a source of cells for cell culture.